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• W2036454611 abstract "We observed that 14 biologically metallated mutants of copper/zinc superoxide dismutase (SOD1) associated with familial amyotrophic lateral sclerosis all exhibited aberrantly accelerated mobility during partially denaturing PAGE and increased sensitivity to proteolytic digestion compared with wild type SOD1. Decreased metal binding site occupancy and exposure to the disulfide-reducing agents dithiothreitol, Tris(2-carboxyethyl)phosphine (TCEP), or reduced glutathione increased the fraction of anomalously migrating mutant SOD1 proteins. Furthermore, the incubation of mutant SOD1s with TCEP increased the accessibility to iodoacetamide of cysteine residues that normally participate in the formation of the intrasubunit disulfide bond (Cys-57 to Cys-146) or are buried within the core of the β-barrel (Cys-6). SOD1 enzymes in spinal cord lysates from G85R and G93A mutant but not wild type SOD1 transgenic mice also exhibited abnormal vulnerability to TCEP, which exposed normally inaccessible cysteine residues to modification by maleimide conjugated to polyethylene glycol. These results implicate SOD1 destabilization under cellular disulfide-reducing conditions at physiological pH and temperature as a shared property that may be relevant to amyotrophic lateral sclerosis mutant neurotoxicity. We observed that 14 biologically metallated mutants of copper/zinc superoxide dismutase (SOD1) associated with familial amyotrophic lateral sclerosis all exhibited aberrantly accelerated mobility during partially denaturing PAGE and increased sensitivity to proteolytic digestion compared with wild type SOD1. Decreased metal binding site occupancy and exposure to the disulfide-reducing agents dithiothreitol, Tris(2-carboxyethyl)phosphine (TCEP), or reduced glutathione increased the fraction of anomalously migrating mutant SOD1 proteins. Furthermore, the incubation of mutant SOD1s with TCEP increased the accessibility to iodoacetamide of cysteine residues that normally participate in the formation of the intrasubunit disulfide bond (Cys-57 to Cys-146) or are buried within the core of the β-barrel (Cys-6). SOD1 enzymes in spinal cord lysates from G85R and G93A mutant but not wild type SOD1 transgenic mice also exhibited abnormal vulnerability to TCEP, which exposed normally inaccessible cysteine residues to modification by maleimide conjugated to polyethylene glycol. These results implicate SOD1 destabilization under cellular disulfide-reducing conditions at physiological pH and temperature as a shared property that may be relevant to amyotrophic lateral sclerosis mutant neurotoxicity. Amyotrophic lateral sclerosis (ALS) 1The abbreviations used are: ALS, amyotrophic lateral sclerosis; DTT, 1,4-dithiothreitol; ESI-MS, electrospray ionization mass spectrometry; GSH, reduced glutathione; Mal-PEG, maleimide conjugated to polyethylene glycol; SOD1, copper/zinc superoxide dismutase; TCEP, Tris(2-carboxyethyl)phosphine; WT, wild type 1The abbreviations used are: ALS, amyotrophic lateral sclerosis; DTT, 1,4-dithiothreitol; ESI-MS, electrospray ionization mass spectrometry; GSH, reduced glutathione; Mal-PEG, maleimide conjugated to polyethylene glycol; SOD1, copper/zinc superoxide dismutase; TCEP, Tris(2-carboxyethyl)phosphine; WT, wild type is an age-dependent degenerative disorder of motor neurons in the spinal cord, brain stem, and brain (1Rowland L.P. Shneider N.A. N. Engl. J. Med. 2001; 344: 1688-1700Google Scholar). Approximately 10% of ALS cases are familial, and ∼20% of these individuals inherit one of >90 autosomal dominant mutations in the gene encoding copper/zinc superoxide dismutase 1 (SOD1) (2Rosen D.R. Siddique T. Patterson D. Figlewicz D.A. Sapp P. Hentati A. Donaldson D. Goto J. O'Regan J.P. Deng H.X. et al.Nature. 1993; 362: 59-62Google Scholar). 2An updated list is posted at www.alsod.org. 2An updated list is posted at www.alsod.org.SOD1 is a 32-kDa homodimeric enzyme expressed predominantly in the cytosol that decreases the intracellular concentration of superoxide radicals (O 2⨪) by catalyzing their dismutation to O2 and H2O2. ALS-associated mutations of conserved residues throughout the protein impart a toxic property to the enzyme that appears unrelated to its normal dismutase activity (reviewed in Ref. 3Cleveland D.W. Rothstein J.D. Nat. Rev. Neurosci. 2001; 2: 806-819Google Scholar). Whereas transgenic mice that overexpress mutant SOD1s consistently develop lethal motor neuron degeneration (4Gurney M.E. Pu H. Chiu A.Y. Dal Canto M.C. Polchow C.Y. Alexander D.D. Caliendo J. Hentati A. Kwon Y.W. Deng H.X. et al.Science. 1994; 264: 1772-1775Google Scholar, 5Wong P.C. Pardo C.A. Borchelt D.R. Lee M.K. Copeland N.G. Jenkins N.A. Sisodia S.S. Cleveland D.W. Price D.L. Neuron. 1995; 14: 1105-1116Google Scholar, 6Ripps M.E. Huntley G.W. Hof P.R. Morrison J.H. Gordon J.W. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 689-693Google Scholar, 7Bruijn L.I. Becher M.W. Lee M.K. Anderson K.L. Jenkins N.A. Copeland N.G. Sisodia S.S. Rothstein J.D. Borchelt D.R. Price D.L. Cleveland D.W. Neuron. 1997; 18: 327-338Google Scholar, 8Dal Canto M.C. Gurney M.E. Acta Neuropathol. (Berlin). 1997; 93: 537-550Google Scholar), mice that overexpress the wild type (WT) enzyme exhibit only subtle motor abnormalities (9Jaarsma D. Haasdijk E.D. Grashorn J.A. Hawkins R. van Duijn W. Verspaget H.W. London J. Holstege J.C. Neurobiol. Dis. 2000; 7: 623-643Google Scholar). In addition, SOD1 knock-out mice are not susceptible to motor neuron loss unless following axonal injury (10Reaume A.G. Elliott J.L. Hoffman E.K. Kowall N.W. Ferrante R.J. Siwek D.F. Wilcox H.M. Flood D.G. Beal M.F. Brown Jr., R.H. Scott R.W. Snider W.D. Nat. Genet. 1996; 13: 43-47Google Scholar).Mutant SOD1 enzymes have been proposed to facilitate aberrant copper-mediated chemistry, disrupt protein recycling or chaperone function, form toxic aggregates, or induce organelle dysfunction or apoptosis (3Cleveland D.W. Rothstein J.D. Nat. Rev. Neurosci. 2001; 2: 806-819Google Scholar, 11Brown Jr., R.H. Curr. Opin. Neurobiol. 1995; 5: 841-846Google Scholar, 12Okado-Matsumoto A. Fridovich I. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 9010-9014Google Scholar), but the precise mechanism of specific motor neuron toxicity has not been elucidated. The observation that some mutant SOD1s exhibit accelerated turnover in vivo or increased proteolytic susceptibility compared with the WT enzyme (13Borchelt D.R. Lee M.K. Slunt H.S. Guarnieri M. Xu Z.S. Wong P.C. Brown Jr., R.H. Price D.L. Sisodia S.S. Cleveland D.W. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 8292-8296Google Scholar, 14Hoffman E.K. Wilcox H.M. Scott R.W. Siman R. J. Neurol. Sci. 1996; 139: 15-20Google Scholar, 15Ratovitski T. Corson L.B. Strain J. Wong P. Cleveland D.W. Culotta V.C. Borchelt D.R. Hum. Mol. Genet. 1999; 8: 1451-1460Google Scholar) suggests that biologically significant perturbations of mutant SOD1 conformation occur. The induction of chaperone proteins that can protect cultured motor neurons from mutant SOD1 toxicity (16Bruening W. Roy J. Giasson B. Figlewicz D.A. Mushynski W.E. Durham H.D. J. Neurochem. 1999; 72: 693-699Google Scholar) and appear to associate with SOD1 mutants (17Shinder G.A. Lacourse M.C. Minotti S. Durham H.D. J. Biol. Chem. 2001; 276: 12791-12796Google Scholar) provides further evidence that destabilization or unfolding of mutant SOD1s in vivomay be related to their toxicity.We previously purified 14 different biologically metallated ALS mutant SOD1s and observed that one group of “WT-like” mutants (A4V, L38V, G41S, G72S, D76Y, D90A, G93A, and E133Δ) bound copper in a fully active coordination environment remarkably similar to that of normal SOD1 despite causing a lethal phenotype (18Hayward L.J. Rodriguez J.A. Kim J.W. Tiwari A. Goto J.J. Cabelli D.E. Valentine J.S. Brown Jr., R.H. J. Biol. Chem. 2002; 277: 15923-15931Google Scholar). The other six “metal-binding region” mutants (H46R, H48Q, G85R, D124V, D125H, and S134N) were clearly distinguished from WT SOD1 according to decreased metal ion contents, altered visible absorption spectra, or decreased specific activities. In a further analysis by differential scanning calorimetry, we found that metal-binding region mutants generally exhibited a larger fraction of species that unfolded at lowT m values compared with those of the WT-like mutants and normal SOD1 (19Rodriguez J.A. Valentine J.S. Eggers D.K. Roe J.A. Tiwari A. Brown Jr., R.H. Hayward L.J. J. Biol. Chem. 2002; 277: 15932-15937Google Scholar). Although purified SOD1 mutants can lose in vitro metal ion binding specificity following partial denaturation at non-physiological pH (20Goto J.J. Zhu H. Sanchez R.J. Nersissian A. Gralla E.B. Valentine J.S. Cabelli D.E. J. Biol. Chem. 2000; 275: 1007-1014Google Scholar), the retention of many native properties in the “as-isolated” WT-like mutants suggests that other influences may destabilize these SOD1 mutants in vivo.Fully metallated bovine SOD1 is active in 4% SDS or 8 murea (21Forman H.J. Fridovich I. J. Biol. Chem. 1973; 248: 2645-2649Google Scholar) and melts in solution at temperatures above 90 °C (22Roe J.A. Butler A. Scholler D.M. Valentine J.S. Marky L. Breslauer K.J. Biochemistry. 1988; 27: 950-958Google Scholar). SOD1 also retains its dimeric quaternary structure upon exposure to 1% SDS in the absence of other denaturing stresses such as heat, urea, reducing agents, or EDTA (23Keele Jr., B.B. McCord J.M. Fridovich I. J. Biol. Chem. 1971; 246: 2875-2880Google Scholar, 24Abernethy J.L. Steinman H.M. Hill R.L. J. Biol. Chem. 1974; 249: 7339-7347Google Scholar, 25Malinowski D.P. Fridovich I. Biochemistry. 1979; 18: 5055-5060Google Scholar). Structural properties of SOD1 that contribute to its extreme thermochemical stability include an eight-stranded β-barrel motif, binding sites for copper and zinc ions, hydrophobic interactions associated with dimerization, and an unusual intrasubunit disulfide bond bridging a loop residue, Cys-57, and Cys-146 of the β-barrel (24Abernethy J.L. Steinman H.M. Hill R.L. J. Biol. Chem. 1974; 249: 7339-7347Google Scholar, 26Bertini I. Mangani S. Viezzoli M.S. Adv. Inorg. Chem. 1998; 45: 127-250Google Scholar). The loop that includes Cys-57 also strongly influences the conformation of Arg-143, which regulates steering of superoxide anion and reactivity of the copper ion via a local hydrogen bond network and the disulfide linkage to Cys-146 (27Banci L. Benedetto M. Bertini I. Del Conte R. Piccioli M. Viezzoli M.S. Biochemistry. 1998; 37: 11780-11791Google Scholar, 28Banci L. Bertini I. Del Conte R. Fadin R. Mangani S. Viezzoli M.S. J. Biol. Inorg. Chem. 1999; 4: 795-803Google Scholar, 29Banci L. Bertini I. Cramaro F. Del Conte R. Viezzoli M.S. Eur. J. Biochem. 2002; 269: 1905-1915Google Scholar). Furthermore, portions of this loop contribute to the dimer interface and form the zinc ion binding site (26Bertini I. Mangani S. Viezzoli M.S. Adv. Inorg. Chem. 1998; 45: 127-250Google Scholar). Conservation of these structural features in all eukaryotic SOD1s suggests that conformational stability of the enzyme, in general, and the disulfide loop, in particular, is critical under physiological conditions.The ratio of reduced (GSH) to oxidized glutathione ([GSH]/[oxidized glutathione]) of ∼30−100:1 in the cytosol (30Freedman R.B. Cell. 1989; 57: 1069-1072Google Scholar) normally inhibits the formation of disulfide linkages in cytosolic proteins (31Raina S. Missiakas D. Annu. Rev. Microbiol. 1997; 51: 179-202Google Scholar), yet WT SOD1 maintains a strong disulfide bond. We hypothesized that the disulfide linkage in ALS-related SOD1 mutants could be vulnerable to cleavage under cellular reducing conditions, which might further destabilize even WT-like mutants. In this study, we correlated changes in electrophoretic mobility, cysteine accessibility to modifying reagents, and protease susceptibility upon incubation of purified SOD1 mutants with disulfide-reducing agents. We then compared sulfhydryl accessibility of WT versus mutant SOD1 proteins in tissue lysates from transgenic mice expressing WT, G85R, or G93A SOD1s under reducing conditions. Our findings suggest that SOD1 destabilization related to thiol-reducing influences in the spinal cord and brain may contribute to the toxicity of mutant SOD1 enzymes in familial ALS.DISCUSSIONThe conformation and stability of ALS-related SOD1 mutants in affected tissues may be influenced directly by the mutant substitutions or indirectly as a consequence of disulfide bond reduction, decreased metal ion binding, monomerization, or other vulnerabilities under conditions in vivo. In this study, we correlated the effects of disulfide-reducing agents upon electrophoretic mobility, cysteine accessibility, and protease sensitivity among purified WT and ALS-related SOD1 variants of known metal ion content (18Hayward L.J. Rodriguez J.A. Kim J.W. Tiwari A. Goto J.J. Cabelli D.E. Valentine J.S. Brown Jr., R.H. J. Biol. Chem. 2002; 277: 15923-15931Google Scholar). We further demonstrated that G85R and G93A mutant SOD1s in transgenic mouse tissue lysates were susceptible to modification at normally inaccessible cysteine residues under reducing conditions.Our initial observation that all 14 of the ALS mutants exhibited accelerated migration during partially denaturing PAGE (Figs. Figure 1, Figure 2, Figure 3) indicated that ALS mutants share properties distinct from WT SOD1. To clarify the nature of those properties, we showed that the mutant proteins were more susceptible than WT SOD1 to disulfide reduction by TCEP (Fig. 5) and proteolysis in the presence of DTT (Fig. 6). Metal-binding region SOD1 mutants (H46R, G85R, D124V, D125H, and S134N) that were severely deficient in copper and zinc ions (18Hayward L.J. Rodriguez J.A. Kim J.W. Tiwari A. Goto J.J. Cabelli D.E. Valentine J.S. Brown Jr., R.H. J. Biol. Chem. 2002; 277: 15923-15931Google Scholar) exhibited the greatest increase in PAGE mobility (Fig. 1), increased reactivity to iodoacetamide in the presence of TCEP (Fig. 5), and greater susceptibility to proteolysis (Fig. 6). This behavior was consistent with the known importance of metal ion binding to SOD1 stability. However, even WT-like mutants (A4V, L38V, G41S, G72S, D76Y, D90A, G93A, and E133Δ), which had more normal metal ion coordination and specific dismutase activity (18Hayward L.J. Rodriguez J.A. Kim J.W. Tiwari A. Goto J.J. Cabelli D.E. Valentine J.S. Brown Jr., R.H. J. Biol. Chem. 2002; 277: 15923-15931Google Scholar), could be distinguished from WT SOD1 under disulfide-reducing conditions (Figs. 1 and 5).The large loop containing residues 49–84, which is anchored by the disulfide bond to the β-barrel, not only composes part of the dimer interface but also forms the zinc binding site that links directly to the copper ion via His-63 (44Tainer J.A. Getzoff E.D. Beem K.M. Richardson J.S. Richardson D.C. J. Mol. Biol. 1982; 160: 181-217Google Scholar). In addition, the backbone oxygen atoms of residues Cys-57 and Gly-61 in this loop form three hydrogen bonds that correctly orient the side chain of Arg-143, an important determinant of copper ion accessibility in SOD1 (27Banci L. Benedetto M. Bertini I. Del Conte R. Piccioli M. Viezzoli M.S. Biochemistry. 1998; 37: 11780-11791Google Scholar, 28Banci L. Bertini I. Del Conte R. Fadin R. Mangani S. Viezzoli M.S. J. Biol. Inorg. Chem. 1999; 4: 795-803Google Scholar, 45Beyer Jr., W.F. Fridovich I. Mullenbach G.T. Hallewell R. J. Biol. Chem. 1987; 262: 11182-11187Google Scholar, 46Fisher C.L. Cabelli D.E. Tainer J.A. Hallewell R.A. Getzoff E.D. Proteins. 1994; 19: 24-34Google Scholar, 47Banci L. Bertini I. Cramaro F. Del Conte R. Rosato A. Viezzoli M.S. Biochemistry. 2000; 39: 9108-9118Google Scholar, 48Lamb A.L. Torres A.S. O'Halloran T.V. Rosenzweig A.C. Nat. Struct. Biol. 2001; 8: 751-755Google Scholar). These structural features increase the likelihood that disulfide reduction at Cys-57, and the disorder of this loop could facilitate partial unfolding, monomerization, metal ion loss, or altered reactivity of bound copper. Human SOD1 also contains two cysteine residues (Cys-6 and Cys-111) that increase the irreversibility of SOD1 thermal unfolding (49Lepock J.R. Frey H.E. Hallewell R.A. J. Biol. Chem. 1990; 265: 21612-21618Google Scholar). The buried side chain of Cys-6, packed tightly within the interior of the β-barrel, should remain inaccessible to solvent unless the SOD1 core is severely disrupted. For example, sulfhydryl reactivity of Cys-6 in bovine SOD1 occurs during exposure to 6m guanidinium chloride but not 8 m urea (25Malinowski D.P. Fridovich I. Biochemistry. 1979; 18: 5055-5060Google Scholar). In contrast, the side chain of Cys-111 is exposed on the protein surface near the dimer interface and may be conjugated to GSH in vivo (50Schinina M.E. Carlini P. Polticelli F. Zappacosta F. Bossa F. Calabrese L. Eur. J. Biochem. 1996; 237: 433-439Google Scholar). Our mass spectrometry results (Fig. 5) suggest that increased susceptibility to disulfide reduction of SOD1 mutants also contributed to partial unfolding of the β-barrel as indicated by accessibility of all four cysteine residues to modification by iodoacetamide. Consistent with these findings, Mal-PEG labeling of SOD1 at multiple cysteine residues was observed in tissue lysates from G85R and G93A but not WT SOD1 transgenic mice (Fig. 7).SOD1 is unusual in its ability to form a stable intrasubunit disulfide bond in the reducing environment of the cytosol, and this property may also be important for proper folding of the enzyme (51Derman A.I. Prinz W.A. Belin D. Beckwith J. Science. 1993; 262: 1744-1747Google Scholar, 52Battistoni A. Mazzetti A.P. Rotilio G. FEBS Lett. 1999; 443: 313-316Google Scholar). We demonstrated that ALS-related SOD1 mutants were sensitive to physiological concentrations of glutathione (Fig. 3), the main cytosolic thiol-disulfide redox buffer. It is also possible that thiol-disulfide oxidoreductases of the thioredoxin and glutaredoxin pathways (53Prinz W.A. Aslund F. Holmgren A. Beckwith J. J. Biol. Chem. 1997; 272: 15661-15667Google Scholar) could impair disulfide formation or stability during folding of the mutant SOD1 enzymes. In this regard, thioredoxin is up-regulated in erythrocytes of familial ALS patients (54Ogawa Y. Kosaka H. Nakanishi T. Shimizu A. Ohoi N. Shouji H. Yanagihara T. Sakoda S. Biochem. Biophys. Res. Commun. 1997; 241: 251-257Google Scholar), and its transcript is also increased ∼6-fold in ALS spinal cord tissue (55Malaspina A. Kaushik N. de Belleroche J. J. Neurochem. 2001; 77: 132-145Google Scholar), possibly as a defense against oxidative stress. The requirement to maintain cellular redox homeostasis and prevent the formation of non-native disulfide bonds thus might strongly influence the degree of mutant SOD1 misfolding or unfolding in specific tissues.How might the susceptibility of mutant SOD1 enzymes to disulfide reduction relate to motor neuron toxicity in familial ALS? One possibility is that destabilization of the zinc-binding loop upon disulfide cleavage could promote selective zinc ion loss while retaining reactive copper ions at the active site. Zinc-deficient, copper-containing SOD1 is neurotoxic in vitro, possibly by a mechanism involving nitric oxide (56Estevez A.G. Crow J.P. Sampson J.B. Reiter C. Zhuang Y. Richardson G.J. Tarpey M.M. Barbeito L. Beckman J.S. Science. 1999; 286: 2498-2500Google Scholar). Alternatively, disulfide reduction could alter copper ion reactivity by perturbing the position of Arg-143, which influences substrate selectivity or by creating a novel copper binding site upon exposure of free cysteine residues. On the other hand, disease progression in mutant SOD1 transgenic mice is not affected by genetic ablation of copper chaperone-dependent copper loading into SOD1 (57Subramaniam J.R. Lyons W.E. Liu J. Bartnikas T.B. Rothstein J. Price D.L. Cleveland D.W. Gitlin J.D. Wong P.C. Nat. Neurosci. 2002; 5: 301-307Google Scholar) or by expression of SOD1 mutants that severely disrupt the copper binding site (58Wang J. Xu G. Gonzales V. Coonfield M. Fromholt D. Copeland N.G. Jenkins N.A. Borchelt D.R. Neurobiol. Dis. 2002; 10: 128-138Google Scholar). However, the toxicity of bound copper has not been completely excluded because unshielded copper ions may be toxic at even nanomolar concentrations (59Williams R.J.P. da Silva J.J.R.F. Coord. Chem. Rev. 2000; 202: 247-348Google Scholar).Disulfide reduction of the ALS mutants may also contribute to toxicity by mechanisms independent of abnormal copper reactivity. For example, the exposure of hydrophobic residues or reactive cysteines could favor abnormal interactions of SOD1 with itself or with other cellular constituents. Mutant SOD1 can form insoluble protein complexes at early stages in G93A SOD1 transgenic mouse tissues and following proteasome inhibition in cultured cells (60Johnston J.A. Dalton M.J. Gurney M.E. Kopito R.R. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 12571-12576Google Scholar). Similarly, a fraction of total mutant SOD1 in brain and spinal cord tissues from SOD1 mice forms high molecular weight complexes that accumulate with disease progression (61Wang J. Xu G. Borchelt D.R. Neurobiol. Dis. 2002; 9: 139-148Google Scholar) and mouse tissues can exhibit thioflavin-S-positive inclusions (58Wang J. Xu G. Gonzales V. Coonfield M. Fromholt D. Copeland N.G. Jenkins N.A. Borchelt D.R. Neurobiol. Dis. 2002; 10: 128-138Google Scholar). Oxidative stress may also be linked to the accumulation of mutant SOD1 either by decreased proteasome activity or by impaired degradation of oxidatively damaged SOD1 (62Oeda T. Shimohama S. Kitagawa N. Kohno R. Imura T. Shibasaki H. Ishii N. Hum. Mol. Genet. 2001; 10: 2013-2023Google Scholar). An increased burden of partially unfolded SOD1 proteins or complexes could ultimately impair cellular chaperone capacity (16Bruening W. Roy J. Giasson B. Figlewicz D.A. Mushynski W.E. Durham H.D. J. Neurochem. 1999; 72: 693-699Google Scholar), perturb mitochondrial function (63Kong J. Xu Z. J. Neurosci. 1998; 18: 3241-3250Google Scholar), sequester anti-apoptotic factors (12Okado-Matsumoto A. Fridovich I. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 9010-9014Google Scholar), disrupt protein recycling, or impose an unsustainable metabolic cost to vulnerable tissues (reviewed in Ref.3Cleveland D.W. Rothstein J.D. Nat. Rev. Neurosci. 2001; 2: 806-819Google Scholar).Our data suggest that cellular disulfide reducing influences at physiological temperature and pH are sufficient to convert relatively well folded WT-like SOD1 mutants (18Hayward L.J. Rodriguez J.A. Kim J.W. Tiwari A. Goto J.J. Cabelli D.E. Valentine J.S. Brown Jr., R.H. J. Biol. Chem. 2002; 277: 15923-15931Google Scholar) or less stable metal-binding region mutants (19Rodriguez J.A. Valentine J.S. Eggers D.K. Roe J.A. Tiwari A. Brown Jr., R.H. Hayward L.J. J. Biol. Chem. 2002; 277: 15932-15937Google Scholar) into more severely destabilized species. These non-native mutant forms might resemble the subset of highly unstable SOD1 C-terminal truncation mutants (15Ratovitski T. Corson L.B. Strain J. Wong P. Cleveland D.W. Culotta V.C. Borchelt D.R. Hum. Mol. Genet. 1999; 8: 1451-1460Google Scholar, 64Zu J.S. Deng H.X. Lo T.P. Mitsumoto H. Ahmed M.S. Hung W.Y. Cai Z.J. Tainer J.A. Siddique T. Neurogenetics. 1997; 1: 65-71Google Scholar), which also lack the disulfide bond consequent to deletion of Cys-146. Overall, these results implicate susceptibility to conformational destabilization of SOD1 by a cellular reducing environment as a shared property that may be relevant to ALS mutant neurotoxicity. Amyotrophic lateral sclerosis (ALS) 1The abbreviations used are: ALS, amyotrophic lateral sclerosis; DTT, 1,4-dithiothreitol; ESI-MS, electrospray ionization mass spectrometry; GSH, reduced glutathione; Mal-PEG, maleimide conjugated to polyethylene glycol; SOD1, copper/zinc superoxide dismutase; TCEP, Tris(2-carboxyethyl)phosphine; WT, wild type 1The abbreviations used are: ALS, amyotrophic lateral sclerosis; DTT, 1,4-dithiothreitol; ESI-MS, electrospray ionization mass spectrometry; GSH, reduced glutathione; Mal-PEG, maleimide conjugated to polyethylene glycol; SOD1, copper/zinc superoxide dismutase; TCEP, Tris(2-carboxyethyl)phosphine; WT, wild type is an age-dependent degenerative disorder of motor neurons in the spinal cord, brain stem, and brain (1Rowland L.P. Shneider N.A. N. Engl. J. Med. 2001; 344: 1688-1700Google Scholar). Approximately 10% of ALS cases are familial, and ∼20% of these individuals inherit one of >90 autosomal dominant mutations in the gene encoding copper/zinc superoxide dismutase 1 (SOD1) (2Rosen D.R. Siddique T. Patterson D. Figlewicz D.A. Sapp P. Hentati A. Donaldson D. Goto J. O'Regan J.P. Deng H.X. et al.Nature. 1993; 362: 59-62Google Scholar). 2An updated list is posted at www.alsod.org. 2An updated list is posted at www.alsod.org. SOD1 is a 32-kDa homodimeric enzyme expressed predominantly in the cytosol that decreases the intracellular concentration of superoxide radicals (O 2⨪) by catalyzing their dismutation to O2 and H2O2. ALS-associated mutations of conserved residues throughout the protein impart a toxic property to the enzyme that appears unrelated to its normal dismutase activity (reviewed in Ref. 3Cleveland D.W. Rothstein J.D. Nat. Rev. Neurosci. 2001; 2: 806-819Google Scholar). Whereas transgenic mice that overexpress mutant SOD1s consistently develop lethal motor neuron degeneration (4Gurney M.E. Pu H. Chiu A.Y. Dal Canto M.C. Polchow C.Y. Alexander D.D. Caliendo J. Hentati A. Kwon Y.W. Deng H.X. et al.Science. 1994; 264: 1772-1775Google Scholar, 5Wong P.C. Pardo C.A. Borchelt D.R. Lee M.K. Copeland N.G. Jenkins N.A. Sisodia S.S. Cleveland D.W. Price D.L. Neuron. 1995; 14: 1105-1116Google Scholar, 6Ripps M.E. Huntley G.W. Hof P.R. Morrison J.H. Gordon J.W. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 689-693Google Scholar, 7Bruijn L.I. Becher M.W. Lee M.K. Anderson K.L. Jenkins N.A. Copeland N.G. Sisodia S.S. Rothstein J.D. Borchelt D.R. Price D.L. Cleveland D.W. Neuron. 1997; 18: 327-338Google Scholar, 8Dal Canto M.C. Gurney M.E. Acta Neuropathol. (Berlin). 1997; 93: 537-550Google Scholar), mice that overexpress the wild type (WT) enzyme exhibit only subtle motor abnormalities (9Jaarsma D. Haasdijk E.D. Grashorn J.A. Hawkins R. van Duijn W. Verspaget H.W. London J. Holstege J.C. Neurobiol. Dis. 2000; 7: 623-643Google Scholar). In addition, SOD1 knock-out mice are not susceptible to motor neuron loss unless following axonal injury (10Reaume A.G. Elliott J.L. Hoffman E.K. Kowall N.W. Ferrante R.J. Siwek D.F. Wilcox H.M. Flood D.G. Beal M.F. Brown Jr., R.H. Scott R.W. Snider W.D. Nat. Genet. 1996; 13: 43-47Google Scholar). Mutant SOD1 enzymes have been proposed to facilitate aberrant copper-mediated chemistry, disrupt protein recycling or chaperone function, form toxic aggregates, or induce organelle dysfunction or apoptosis (3Cleveland D.W. Rothstein J.D. Nat. Rev. Neurosci. 2001; 2: 806-819Google Scholar, 11Brown Jr., R.H. Curr. Opin. Neurobiol. 1995; 5: 841-846Google Scholar, 12Okado-Matsumoto A. Fridovich I. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 9010-9014Google Scholar), but the precise mechanism of specific motor neuron toxicity has not been elucidated. The observation that some mutant SOD1s exhibit accelerated turnover in vivo or increased proteolytic susceptibility compared with the WT enzyme (13Borchelt D.R. Lee M.K. Slunt H.S. Guarnieri M. Xu Z.S. Wong P.C. Brown Jr., R.H. Price D.L. Sisodia S.S. Cleveland D.W. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 8292-8296Google Scholar, 14Hoffman E.K. Wilcox H.M. Scott R.W. Siman R. J. Neurol. Sci. 1996; 139: 15-20Google Scholar, 15Ratovitski T. Corson L.B. Strain J. Wong P. Cleveland D.W. Culotta V.C. Borchelt D.R. Hum. Mol. Genet. 1999; 8: 1451-1460Google Scholar) suggests that biologically significant perturbations of mutant SOD1 conformation occur. The induction of chaperone proteins that can protect cultured motor neurons from mutant SOD1 toxicity (16Bruening W. Roy J. Giasson B. Figlewicz D.A. Mushynski W.E. Durham H.D. J. Neurochem. 1999; 72: 693-699Google Scholar) and appear to associate with SOD1 mutants (17Shinder G.A. Lacourse M.C. Minotti S. Durham H.D. J. Biol. Chem. 2001; 276: 12791-12796Google Scholar) provides further evidence that destabilization or unfolding of mutant SOD1s in vivomay be related to their toxicity. We previously purified 14 different biologically metallated ALS mutant SOD1s and observed that one group of “WT-like” mutants (A4V, L38V, G41S, G72S, D76Y, D90A, G93A, and E133Δ) bound copper in a fully active coordination environment remarkably similar to that of normal SOD1 despite causing a lethal phenotype (18Hayward L.J. Rodriguez J.A. Kim J.W. Tiwari A. Goto J.J. Cabelli D.E. Valentine J.S. Brown Jr., R.H. J. Biol. Chem. 2002; 277: 15923-15931Google Scholar). The other six “metal-binding region” mutants (H46R, H48Q, G85R, D124V, D125H, and S134N) were clearly distinguished from WT SOD1 according to decreased metal ion contents, altered visible absorption spectra, or decreased specific activities. In a further analysis by differential scanning calorimetry, we found that metal-binding region mutants generally exhibited a larger fraction of species that unfolded at lowT m values compared with those of the WT-like mutants and normal SOD1 (19Rodriguez J.A. Valentine J.S. Eggers D.K. Roe J.A. Tiwari A. Brown Jr., R.H. Hayward L.J. J. Biol. Chem. 2002; 277: 15932-15937Google Scholar). Although purified SOD1 mutants can lose in vitro metal ion binding specificity following partial denaturation at non-physiological pH (20Goto J.J. Zhu H. Sanchez R.J. Nersissian A. Gralla E.B. Valentine J.S. Cabelli D.E. J. Biol. Chem. 2000; 275: 1007-1014Google Scholar), the retention of many native properties in the “as-isolated” WT-like mutants suggests that other influences may destabilize" @default.
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• W2036454611 title "Familial Amyotrophic Lateral Sclerosis Mutants of Copper/Zinc Superoxide Dismutase Are Susceptible to Disulfide Reduction" @default.
• W2036454611 cites W1521574867 @default.
• W2036454611 cites W1523787450 @default.
• W2036454611 cites W1546589453 @default.
• W2036454611 cites W1581159509 @default.
• W2036454611 cites W1585758755 @default.
• W2036454611 cites W1605533832 @default.
• W2036454611 cites W1659903845 @default.
• W2036454611 cites W1697966803 @default.
• W2036454611 cites W1974835781 @default.
• W2036454611 cites W1983959956 @default.
• W2036454611 cites W1985655958 @default.
• W2036454611 cites W1988556092 @default.
• W2036454611 cites W1988849173 @default.
• W2036454611 cites W1990502481 @default.
• W2036454611 cites W1990602502 @default.
• W2036454611 cites W2002500792 @default.
• W2036454611 cites W2003733797 @default.
• W2036454611 cites W2008069164 @default.
• W2036454611 cites W2008874237 @default.
• W2036454611 cites W2012851706 @default.
• W2036454611 cites W2014730205 @default.
• W2036454611 cites W2020250772 @default.
• W2036454611 cites W2021594222 @default.
• W2036454611 cites W2022367303 @default.
• W2036454611 cites W2025153717 @default.
• W2036454611 cites W2027426714 @default.
• W2036454611 cites W2028317073 @default.
• W2036454611 cites W2029684104 @default.
• W2036454611 cites W2030066705 @default.
• W2036454611 cites W2031751971 @default.
• W2036454611 cites W2032107666 @default.
• W2036454611 cites W2033255695 @default.
• W2036454611 cites W2034084833 @default.
• W2036454611 cites W2037365087 @default.
• W2036454611 cites W2038941603 @default.
• W2036454611 cites W2042870632 @default.
• W2036454611 cites W2047687145 @default.
• W2036454611 cites W2050417334 @default.
• W2036454611 cites W2052979701 @default.
• W2036454611 cites W2056789450 @default.
• W2036454611 cites W2068861782 @default.
• W2036454611 cites W2070243481 @default.
• W2036454611 cites W2072978915 @default.
• W2036454611 cites W2073864172 @default.
• W2036454611 cites W2077135798 @default.
• W2036454611 cites W2085880812 @default.
• W2036454611 cites W2094608970 @default.
• W2036454611 cites W2096386031 @default.
• W2036454611 cites W2097929984 @default.
• W2036454611 cites W2099040799 @default.
• W2036454611 cites W2104017463 @default.
• W2036454611 cites W2106392233 @default.
• W2036454611 cites W2107768418 @default.
• W2036454611 cites W2110580010 @default.
• W2036454611 cites W2112796828 @default.
• W2036454611 cites W2113777884 @default.
• W2036454611 cites W2124084307 @default.
• W2036454611 cites W2125501179 @default.
• W2036454611 cites W2125606478 @default.
• W2036454611 cites W2128736563 @default.
• W2036454611 cites W2133408527 @default.
• W2036454611 cites W2169714384 @default.
• W2036454611 cites W4232534952 @default.
• W2036454611 cites W4236884331 @default.
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